This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Fluoroacetate dehalogenases catalyze the hydrolysis of fluoroacetate into glycolate. They possess the rare ability of breaking the carbon-fluorine bond the strongest bond in organic chemistry. They are thus promising candidates in the bioremediation of some of the most persistent environmental contaminants. We are interested in understanding how the defluorinases function at the atomic level to gain valuable insights for developing solutions for degrading fluorinated organic pollutants. Traditionally the structural characterization of enzymatic function or structural enzymology is performed using various trapping strategies. While countless reaction mechanisms have been elucidated by such means these methods suffer from potentially introducing artifacts such as non-physiologically relevant binding modes in mutant enzymes. Therefore we propose to use time-resolved crystallography (TRX) to study the enzymatic cleavage of the C-F bond;TRX is a very powerful tool in structural enzymology because it does not involve any trapping of reaction intermediates. The experiments may be performed using wild-type protein crystals on natural substrates at ambient temperatures and the structures of the reaction intermediates as well as the kinetic models may be simultaneously extracted. More significantly it has the capability of identifying some extremely short-lived structural intermediates (sub-ns timescale) which is not easily achievable by other means. Despite these advantages TRX is not readily applicable in structural enzymology. Firstly a fast method of triggering the reaction (often involving a laser pulse) must be available;this is by far the biggest limitation of TRX. For studying irreversible reactions the requirements become even more demanding because the relatively slow read-out times of current CCD detectors (~ 3 s) would necessitate thousands of protein crystals. Obviously this is prohibitively excessive from the practical point of view. Fortunately a novel data collection strategy developed by Dr. Zhong Ren (BioCARS APS) promises to reduce this requirement by an order of magnitude. Based on our years of experience from working with fluoroacetate dehalogenases it became clear that they are particularly well-suited for TRX: a fast light-trigger can be readily incorporated;the crystallized enzyme is active;the reaction timescales are compatible with the novel data collection strategy;crystals of very high diffraction quality (<1 A) are readily reproducible. We believe that the findings in this work will be highly significant towards developing and establishing this novel TRX strategy as a general approach for studying irreversible systems. At the same time it deals with a problem of great environmental significance.